Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction.

Calpain activation contributes to the development of infection-induced diaphragm weakness, but the mechanisms by which infections activate calpain are poorly understood. We postulated that skeletal muscle calcium-dependent phospholipase A2 (cPLA2) is activated by cytokines and has downstream effects that induce calpain activation and muscle weakness. We determined whether cPLA2 activation mediates cytokine-induced calpain activation in isolated skeletal muscle (C2C12) cells and infection-induced diaphragm weakness in mice. C2C12 cells were treated with the following: 1) vehicle; 2) cytomix (TNF-α 20 ng/ml, IL-1ß 50 U/ml, IFN-γ 100 U/ml, LPS 10 µg/ml); 3) cytomix + AACOCF3, a cPLA2 inhibitor (10 µM); or 4) AACOCF3 alone. At 24 h, we assessed cell cPLA2 activity, mitochondrial superoxide generation, calpain activity, and calpastatin activity. We also determined if SS31 (10 µg/ml), a mitochondrial superoxide scavenger, reduced cytomix-mediated calpain activation. Finally, we determined if CDIBA (10 µM), a cPLA2 inhibitor, reduced diaphragm dysfunction due to cecal ligation puncture in mice. Cytomix increased C2C12 cell cPLA2 activity (P < 0.001) and superoxide generation; AACOCF3 and SS31 blocked increases in superoxide generation (P < 0.001). Cytomix also activated calpain (P < 0.001) and inactivated calpastatin (P < 0.01); both AACOCF3 and SS31 prevented these changes. Cecal ligation puncture reduced diaphragm force in mice, and CDIBA prevented this reduction (P < 0.001). cPLA2 modulates cytokine-induced calpain activation in cells and infection-induced diaphragm weakness in animals. We speculate that therapies that inhibit cPLA2 may prevent diaphragm weakness in infected, critically ill patients.

Efficacy and safety of endometrial ablation for treating abnormal uterine bleeding in pre- and postmenopausal women with liver cirrhosis.

AIM: Abnormal uterine bleeding (AUB) occurs in 10-30% of women of reproductive age and up to 61% of cirrhotic women. We evaluated the efficacy and safety of endometrial ablation (NovaSure therapy) for AUB in cirrhotic women. METHODS: This prospective, two-arm, observational study enrolled patients for NovaSure treatment, and they were followed for 12 months. Primary measurements were the amenorrhea rate and changes of pictorial blood loss assessment chart (PBLAC) scores at 1-month post-therapy. Key secondary end-points included the longevity of amenorrhea at 12 months, safety profile, and progression of cirrhosis. RESULTS: Among 88 women, 26 were cirrhotic and 62 were non-cirrhotic. At 1-month post-NovaSure treatment, a significant reduction of mean PBLAC scores was observed in cirrhotic patients compared to those at baseline (0.4 ± 1.3 vs 215.2 ± 410.9, P < 0.001), and the amenorrhea rate was 88.5%. The efficacy outcomes of the PBLAC scores and amenorrhea rate were maintained until the end of the 12-month follow-up. A significant improvement in quality of life scores was observed 1-month post-therapy compared to those at baseline (5.4 ± 3.1 vs 20.5 ± 5.5, P < 0.001). Patients' satisfaction rates were 100% and 92.31% at 6 and 12 months, respectively. The aforementioned outcomes were comparable with those in non-cirrhotic patients. No significant progression of cirrhosis or safety concern was reported. CONCLUSION: Cirrhotic patients on NovaSure therapy had a high rate of amenorrhea 1-month post-treatment, which maintained longevity for 12 months. The safety profile was similar to that in non-cirrhotic patients.

[Application of high performance liquid-ion trap mass spectrometry in analyzing saponins in sodium aescinate].

Sodium aescinate, which is produced from saponins of Chinese Buckeye Seed, is a prescription drug for treatment of brain edema and all kinds of swellings caused by surgery. In this article, high-performance liquid chromatography/ion trap (HPLC-IT) mass spectrometry was applied to study the characteristic ions of ten reference substances, namely escin Ⅰa, escin Ⅰb, isoescin Ⅰa, isoescin Ⅰb, aesculiside A, aesculiside B, aesculuside A, escin Ⅳc, escinⅡa and escin Ⅴ, which were isolated from aescinate. Furthermore, 19 saponin compounds were predicted in sodium aescinate, besides the above mentioned reference substances. The study showed that sapogenins in sodium aescinate had two structural types, namely protoaescigenin and barringenol C, and the substituent acetyl, tigloyl or angeloyl was usually located at C-21, C-22 or C-28 position. Among these predicted saponins, their sugar chains were all located at C-3 position consisting of glucose and glucuronide. This study provides experimental data for chemical constituents in sodium aescinate and scientific basis for quality and safety evaluation.

Neutral sphingomyelinase 2 is required for cytokine-induced skeletal muscle calpain activation.

Calpain contributes to infection-induced diaphragm dysfunction but the upstream mechanism(s) responsible for calpain activation are poorly understood. It is known, however, that cytokines activate neutral sphingomyelinase (nSMase) and nSMase has downstream effects with the potential to increase calpain activity. We tested the hypothesis that infection-induced skeletal muscle calpain activation is a consequence of nSMase activation. We administered cytomix (20 ng/ml TNF-α, 50 U/ml IL-1ß, 100 U/ml IFN-γ, 10 µg/ml LPS) to C2C12 muscle cells to simulate the effects of infection in vitro and studied mice undergoing cecal ligation puncture (CLP) as an in vivo model of infection. In cell studies, we assessed sphingomyelinase activity, subcellular calcium levels, and calpain activity and determined the effects of inhibiting sphingomyelinase using chemical (GW4869) and genetic (siRNA to nSMase2 and nSMase3) techniques. We assessed diaphragm force and calpain activity and utilized GW4869 to inhibit sphingomyelinase in mice. Cytomix increased cytosolic and mitochondrial calcium levels in C2C12 cells (P < 0.001); addition of GW4869 blocked these increases (P < 0.001). Cytomix also activated calpain, increasing calpain activity (P < 0.02), and the calpain-mediated cleavage of procaspase 12 (P < 0.001). Procaspase 12 cleavage was attenuated by either GW4869 (P < 0.001), BAPTA-AM (P < 0.001), or siRNA to nSMase2 (P < 0.001) but was unaffected by siRNA to nSMase3. GW4869 prevented CLP-induced diaphragm calpain activation and diaphragm weakness in mice. These data suggest that nSMase2 activation is required for the development of infection-induced diaphragm calpain activation and muscle weakness. As a consequence, therapies that inhibit nSMase2 in patients may prevent infection-induced skeletal muscle dysfunction.

[Clinical observation of posterior polymorphous corneal dystrophy].

OBJECTIVE: To research the clinical features and in vivo confocal microscopic findings of posterior polymorphous corneal dystrophy (PPCD). METHODS: It was a retrospective consecutive case study. Ten patients with PPCD, attended at Optometry Department of Henan Eye Institute from March 2007 to August 2009, were analyzed. All the subjects were examined by slit-lamp, OrbscanII, specular microscopy, HRT3/RCM confocal microscopy. Mann-Whitney U test was used to analysis the data. RESULTS: The age of the patients ranged from 8 to 35 years. Seven eyes of the 4 patients have the vesicular lesions, five eyes of the 5 patients were band lesions and 1 patient had bilateral diffused opacities, this patient also had iridocorneal adhesions with associated papillary ectropion but without glaucoma. In total, 14 eyes of the 10 patients had PPCD. Two eyes had abnormal OrbscanII topography, it showed both anterior and posterior surface protrusion. Specular microscopy exam indicated large cells in size and reduced endothelium density. The mean size of the affected eye was 584 µm(2), the normal eye was 316 µm(2). The difference was statistically significant (U = 0.000, P = 0.002). The density of the endothelium was 1746 cells/mm(2) in affected eye and 3201 cells/mm(2) in normal eye. The difference was also statistically significant (U = 0.000, P = 0.002). In vivo confocal microscopy showed endothelial polymorphism. Occasional bright endothelial nuclei were seen. A variety of curvilinear and vesicular abnormalities were imaged including orange or finger like lesion, round or oval dark area with hyper reflectivity border. Some large lesions may lose endothelium with rough surface have a dike appearance. CONCLUSIONS: Careful exam by slit-lamp may help to diagnose PPCD and further specular microscopy and(or) in vivo confocal microscopy exam will confirm it. Some cases may have abnormal topography, or associated with high intraocular pressure.

The metabolites of mangrove endophytic fungus Zh6-B1 from the South China Sea.

Two new metabolites, 3R,5R-Sonnerlactone (1) and 3R,5S-Sonnerlactone (2), were isolated from the mangrove endophytic fungus Zh6-B1 obtained from the South China Sea. Their structures were elucidated by MS and NMR. The absolute configuration of compound 1 was determined by single-crystal X-ray analysis using Cu Kalpha radiation. The absolute configuration of compound 2 was determined by NOESY analysis and comparing circular dichroism spectroscopy with compound 1. The antiproliferative activity of compound 1 and 2 against the multi-drug resistant human oral floor carcinoma cells (KV) was evaluated.

[Proteasome inhibitors sensitize ovarian cancer cells to cisplatin].

OBJECTIVE: To explore the sensitivity and the molecular mechanism of cisplatin-resistance ovarian cancer cell line C13 to proteasome inhibitors and the combination with cisplatin. METHODS: After different treatments, methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability, annexin-V/propidium iodide (PI) apoptosis detection kit was used to determine the apoptosis rate of different groups, western blot assay was introduced to evaluate the expression levels of Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein (cFLIPs), and the activity of caspase-8 was examined. RESULTS: MTT assay shown that the cell viability ratios of combination group at serial time points from 12, 24, 36, 48, 60, 72 hours were (56.0 ± 8.4) %, (44.7 ± 7.3) %, (33.7 ± 11.2) %, (27.6 ± 8.0) %, (27.6 ± 7.6) % and (28.1 ± 2.4) %, which were much lower than those of cisplatin group (P < 0.05). After treated for 24 hours, apoptosis rates of cisplatin group, bortezomib group and combination group were (16.7 ± 1.7) %, (23.4 ± 2.1) % and (26.9 ± 1.6) %, respectively. The rate of combination group was much higher than that of non-treated group and that of cisplatin group or bortezomib group (P < 0.05). Western blot assay showed the changes of expression levels of cFLIPs, which were down-regulated seriously after cisplatin, bortezomib or combination treatment [(43.2 ± 2.3) % vs (75.7 ± 3.0) % vs (67.9 ± 2.1) %, P < 0.05]. The caspase-8 activity of combination group was (5.6 ± 1.6) folds than that of non-treated group, which was higher than those of other two groups [(2.3 ± 1.0) and (4.2 ± 0.9) folds, P < 0.05]. CONCLUSIONS: The tumor cell lethal effect of cisplatin could be increase significantly by the combination application of proteasome inhibitors, bortezomib. And the cFLIPs/caspase-8 signaling pathway may be play an important role in the molecular mechanism of the combination treatment.

[Relationships between constitutional types of traditional Chinese medicine and hypertension].

OBJECTIVE: To investigate the relationships between constitutional types of traditional Chinese medicine (TCM) and hypertension so as to provide epidemiological evidence for the theory of correlation between constitution and disease. METHODS: A cross-sectional survey of TCM constitution data from a population of 7 782 from Beijng and 8 provinces of China (Jiangsu, Anhui, Gansu, Qinghai, Fujian, Jilin, Jiangxi, and Henan) was made in the study. The survey of TCM constitutions was performed by standardized TCM Constitution Questionnaire. Discriminatory analysis was used to judge the individual constitutional types including normal constitution, and qi deficiency, yang deficiency, yin deficiency, phlegm-dampness, damp-heat, blood stasis, qi stagnation and special constitutions. A multiple stepwise logistic regression analysis was applied to explore the significantly influential constitutional factors of hypertension. RESULTS: After controlling several factors like gender, age, marital status, occupation, and educational background, three TCM constitutional factors according to different degrees of relative risks were entered into the multiple stepwise logistic regression model. The three factors were phlegm-dampness, yin deficiency and qi deficiency constitutions, and the odds ratio (OR) and 95% confidence interval (CI) were 2.00 [1.58, 2.55], 1.66 [1.33, 2.08] and 1.37 [1.13, 1.66] respectively. The main constitutional influential factors of hypertension in male patients were phlegm-dampness and yin deficiency constitutions, with OR and 95% CI of 1.61 [1.22, 2.14] and 1.60 [1.17, 2.19]. Phlegm-dampness, yin deficiency and qi deficiency constitutions were the main constitutional influential factors of hypertension in female patients. The OR and 95% CI were 2.80 [1.79, 4.39], 1.55 [1.13, 2.14] and 1.39 [1.05, 1.84] respectively. Phlegm-dampness constitution had more influence on hypertension in female patients than other constitution types. CONCLUSION: Phlegm-dampness, yin deficiency, and qi deficiency constitutions are the main influential factors of hypertension. Hypertensive patients with different gender have different constitutional influential factors.

Association of Serum Uric Acid Concentration and Its Change with Cardiovascular Death and All-Cause Mortality.

OBJECTIVE: There is no consensus on the role of abnormal uric acid (UA) levels in the prognosis of patients undergoing hemodialysis. We therefore aimed to investigate the effects of changes in UA concentration on the risk of all-cause death and cardiac death in such patients. METHOD: In this retrospective cohort study, patients admitted to two hemodialysis centers performing maintenance hemodialysis (MHD) in Wuhan First Hospital and Fourth Hospital Hemodialysis Center from January 1, 2007, to October 31, 2017, were included. RESULTS: In all, 325 patients undergoing MHD aged 59.7 ± 14.7 years, including 195 men (60%), were enrolled, with a median follow-up of 37 months. Serum UA (p < 0.001) was significantly higher in the surviving group than in the death group. No significant difference was found in UA variability (p < 0.001) was significantly higher in the surviving group than in the death group. No significant difference was found in UA variability (p < 0.001) was significantly higher in the surviving group than in the death group. No significant difference was found in UA variability (p < 0.001) was significantly higher in the surviving group than in the death group. No significant difference was found in UA variability (p < 0.001) was significantly higher in the surviving group than in the death group. No significant difference was found in UA variability (p < 0.001) was significantly higher in the surviving group than in the death group. No significant difference was found in UA variability (p < 0.001) was significantly higher in the surviving group than in the death group. No significant difference was found in UA variability (p < 0.001) was significantly higher in the surviving group than in the death group. No significant difference was found in UA variability (. CONCLUSION: Low UA levels were closely related to all-cause mortality in patients undergoing MHD. Although UA levels had no significant effect on cardiac death, they had a good predictive value for long-term prognosis in patients on MHD.

The circulating level of miR-122 is a potential risk factor for endothelial dysfunction in young patients with essential hypertension.

MicroRNAs are key molecules involved in the regulation of endothelial function. They are important risk factors and biomarkers for the development of hypertension related to endothelial dysfunction. However, the gene expression patterns associated with hypertension development related to endothelial dysfunction have not been fully elucidated. We conducted a case-control study of 65 patients with essential hypertension (EH) and 61 controls without EH. Plasma levels of miR-122 and its target protein high-affinity cationic amino acid transporter 1 (CAT-1) were measured by qRT-PCR and ELISA, respectively. miR-122 expression in plasma of patients with EH was significantly higher than that of the control group (p = 0.001), while CAT-1 expression in patients with EH was significantly lower than that in the control group (p = 0.018). miR-122 expression in plasma of young patients with EH was significantly higher than that in young people without EH (p = 0.0004), and CAT-1 expression in plasma of young patients with EH was also significantly lower than that of the control group (p = 0.002). CAT-1 expression in the plasma of young participants was significantly higher than that of individuals aged ≥40 years (p = 0.003), whereas miR-122 expression was significantly lower (p = 0.001). We showed that among patients with EH, the high expression of miR-122 contributed to endothelial dysfunction by suppressing the expression of the CAT-1 protein, which led to a decrease in CAT-1 expression in plasma. Therefore, high expression of miR-122 appears to be a risk factor for endothelial dysfunction in EH, especially in younger patients.

Androgen-regulated formation and degradation of gap junctions in androgen-responsive human prostate cancer cells.

The constituent proteins of gap junctions, called connexins (Cxs), have a short half-life. Despite this, the physiological stimuli that control the assembly of Cxs into gap junctions and their degradation have remained poorly understood. We show here that in androgen-responsive human prostate cancer cells, androgens control the expression level of Cx32-and hence the extent of gap junction formation-post-translationally. In the absence of androgens, a major fraction of Cx32 is degraded presumably by endoplasmic reticulum-associated degradation, whereas in their presence, this fraction is rescued from degradation. We also show that Cx32 and Cx43 degrade by a similar mechanism. Thus, androgens regulate the formation and degradation of gap junctions by rerouting the pool of Cxs, which normally would have been degraded from the early secretory compartment, to the cell surface, and enhancing assembly into gap junctions. Androgens had no significant effect on the formation and degradation of adherens and tight junction-associated proteins. The findings that in a cell culture model that mimics the progression of human prostate cancer, degradation of Cxs, as well as formation of gap junctions, are androgen-dependent strongly implicate an important role of junctional communication in the prostate morphogenesis and oncogenesis.

Inhibition of cytochrome P450 enzymes by rhein in rat liver microsomes.

Rhein, an active ingredient extensively found in plants such as Aloe, Cassitora L., rhubarb and so on, has been used for a long time in China. Pharmacological tests revealed that rhein not only had a strong antibacterial action, but also may be useful in cancer chemotherapy as a biochemical modulator. Its therapeutic action and toxicity is still the subject of considerable research. With microsome incubation assays in vitro and HPLC methods, the inhibition of rat liver CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A enzymes by rhein were studied kinetically. The results showed the most inhibition of CYP2E1 by rhein (K(i) = 10 microm, mixed); CYP3A and CYP2C9 were also inhibited by rhein, K(i) = 30 microm (mixed) and K(i) = 38 microm (mixed), respectively; rhein revealed some inhibition of CYP1A2 (K(i) = 62 microm, uncompetitive) and CYP2D6 (K(i) = 74 microm, mixed). Drug-drug interactions, especially cytochrome P450 (CYP)-mediated interactions, cause an enhancement or attenuation in the efficacy of co-administered drugs. Inhibition of the five major CYP enzymes observed for rhein suggested that changes in pharmacokinetics of co-administered drugs were likely to occur. Therefore, caution should be paid to the possible drug interaction of medicinal plants containing rhein and CYP substrates.

[Effects of different glucocorticoids on blood glucose during surgery under general anesthesia].

OBJECTIVE: To evaluate the effects of different glucocorticoids upon blood glucose during surgery under general anesthesia. METHODS: Thirty (ASA I/II) patients scheduled for surgery under combined intravenous-inhalational anesthesia were randomly divided into 3 groups: dexamethasone group (Group D), methylprednisolone group (Group M) and normal saline group (Group S). Patients received dexamethasone, 10 mg IV (Group D), or methylprednisolone, 120 mg IV (Group M), or normal saline, 10 ml IV (Group S) at pre-induction. Arterial blood was collected preoperatively, and at 60, 120, 180 min post-injection to analyze the blood glucose. RESULTS: As compared to baseline, blood glucose increased significantly in Group D at 60, 120, 180 min post-injection (P < 0.01). In Group M, blood glucose increased significantly at 180 min post-injection (P < 0.01). In Group S, blood glucose increased significantly at 60, 120, 180 min post-injection respectively (P < 0.01). Comparison among these three groups indicated that differences between Group D and Group M (P = 0.01) or Group S (P < 0.05) were significant at 120 min post-injection. The difference between Group D and Group M was significant at 180 min post-injection (P < 0.05). CONCLUSION: The blood glucose often increases significantly during surgery under general anesthesia. Use of glucocorticoids may aggravate hyperglycemia, so that close monitoring and control are quite important. Methylprednisolone has less effect upon blood glucose than dexamethasone.

[Analysis of Y-chromosomal biallelic polymorphisms in Sichuan Han of Chinese population].

OBJECTIVE: To evaluate the forensic utility of Y-single nucleotide polymorphisms (SNPs) markers. METHODS: Allele-specific PCR, restriction enzyme digestion or direct PCR were performed to examine 10 different SNP loci on Y chromosome, namely M9, M15, M45, M89, M95, M122, M134, M145, M173 and P25 in 161 Chinese Han males. RESULTS: A total of 8 of the 10 SNPs are reported to be polymorphic in Chinese. The gene diversity for the loci showing polymorphism ranged from 0.988/0.012-0.752/0.248, with a power of discrimination 0.094-0.373. Loci M122 and M134 were the most polymorphic markers in Chinese Hans. Nine different haplogroups with frequencies from 1.2% to 51.6% were observed and 3 of the haplogroups-K*(x O2a, O3, P), O3*(x O3e) and O3e were found in 75.2% of Chinese Hans. CONCLUSION: A comprehensive gene diversity data of Y chromosome and haplogroups were obtained in Sichuan Han population, which will be served as the base for using these Y-SNP markers in forensic medicine and individual identification in Sichuan Hans.

[Proteasome inhibitors sensitize ovarian cancer cells to paclitaxel induced apoptosis].

OBJECTIVE: To explore the sensitivity of ovarian cancer cell line SKOV3 to paclitaxel, proteasome inhibitors, bortezomib, and their combination. METHODS: The methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability after treatment. The annexin V-propidium iodide apoptosis detection kit was used to determine the apoptosis rate of different groups. Western blot assay was used to evaluate the expression levels of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK-3beta). RESULTS: In MTT assay, the cell viability ratios of the combination group at serial time points from 12, 24, 36, 48 and 72 hours were (65.2 +/- 5.8)%, (58.3 +/- 14.4)%, (35.3 +/- 5.0)%, (19.2 +/- 1.5)%, and (11.4 +/- 2.5)%, which were significantly lower than those of the paclitaxel group (P < 0.05). After drug treatments, apoptosis rates of paclitaxel group, bortezomib group and the combination group were (14.7 +/- 0.5)%, (15.1 +/- 0.8)% and (20.5 +/- 0.7)% respectively. The rate of the combination group was significantly higher than that of non-treated group and paclitaxel group (P < 0.05). Western blot assay showed the changes in expression levels of phosphorylated AKT and GSK-3beta, which were decreased significantly after paclitaxel and bortezomib combination treatment [(3.2 +/- 0.8)%, (19.3 +/- 0.4)%; P < 0.05]. CONCLUSIONS: The lethal effect of paclitaxel on tumor cells could be increased significantly by its combination with proteasome inhibitors, bortezomib. The AKT/GSK-3beta signaling pathway plays an important role in the molecular mechanism of the combination treatment.

[Study on the accumulation of fluoroquinolones in clinical isolates of susceptible and multi-drug-resistant Escherichia coli strains].

OBJECTIVE: To investigate the active efflux of fluoroquinolones in clinical isolated strains of Escherichia coli. METHODS: The accumulation of ciprofloxacin and tosufloxacin for clinical isolates of Escherichia coli and the effect of glucose and Carbonyl cyanide mchlorophenyl hydrazone (CCCP) on the uptake of Escherichia coli were determined by a modified fluorometric assay. RESULTS: The accumulation of ciprofloxacin in sensitive and multi-drug-resistant (MDR) organisms decreased with energy dependence, and the decrease in MDR isolate was prominent. Incubation with CCCP increased the accumulation of ciprofloxacin up to three-fold in MDR isolate but not in sensitive isolate. In contrast, CCCP appeared to have no influence on the uptake of tosufloxacin in Escherichia coli. CONCLUSION: These results indicate that energy-dependent active efflux is one of the mechanisms of hydrophilic fluoroquinolones resistance, but it does not play any role in hydrophobic fluoroquinolones resistance in Escherichia coli.

[Expression and analysis of biological activity of the recombination anthrax edema factor].

Anthrax toxin consists of three separate proteins, protective antigen (PA), lethal factor (LF) and edema factor (EF). EF is bacterial adenylate cyclase which, upon activation by its eukaryotic cofactor, calmodulin, causes a rapid increase in the intracellular cAMP level of host cells. EF can reduce the protective ability of host animal. In order to further research the mechanism of anthrax toxin, the expression plasmid was constructed and the structural gene for anthrax edema factor (EF) was expressed in Escherichia coli. Recombinant EF (rEF) was purified to homogeneity by a three-step procedure involving metal chelating affinity chromatography, cation-exchange chromatography and gel chromatography. From 1 liter of culture, 5mg of biologically active EF was easily purified. Rabbits were immuned with rEF, anti-EF antibodies were prepared and can neutralize rEF in cells. Tests in vitro proved rEF have good biological activity. rEF can competed the binding regions of PA with rLF in J774A.1 and CHO cells. rEF and rLF can restrain each other by competition. The successful expression of rEF has placed a solid foundation for the research on toxicity mechanism of EF, and screening for inhibitors against EF.

Muscle-specific calpastatin overexpression prevents diaphragm weakness in cecal ligation puncture-induced sepsis.

Recent work indicates that infections are a major contributor to diaphragm weakness in patients who are critically ill and mechanically ventilated, and that diaphragm weakness is a risk factor for death and prolonged mechanical ventilation. Infections activate muscle calpain, but many believe this is an epiphenomenon and that other proteolytic processes are responsible for infection-induced muscle weakness. We tested the hypothesis that muscle-specific overexpression of calpastatin (CalpOX; an endogenous calpain inhibitor) would attenuate diaphragm dysfunction in cecal ligation puncture (CLP)-induced sepsis. We studied 1) wild-type (WT) sham-operated mice, 2) WT CLP-operated mice, 3) CalpOX sham-operated mice, and 4) CalpOX CLP-operated mice (n = 9-10/group). Twenty-four hours after surgery, we assessed the diaphragm force-frequency relationship, diaphragm mass, and total protein content and diaphragm levels of talin and myosin heavy chain (MHC). CLP markedly reduced diaphragm-specific force generation (force/cross-sectional area), which was prevented by calpastatin overexpression (force averaged 21.4 ± 0.5, 6.9 ± 0.8, 22.4 ± 1.0, and 18.3 ± 1.3 N/cm(2), respectively, for WT sham, WT CLP, CalpOX sham, and CalpOX CLP groups, P < 0.001). Diaphragm mass and total protein content were similar in all groups. CLP induced talin cleavage and reduced MHC levels; CalpOX prevented these alterations. CLP-induced sepsis rapidly reduces diaphragm-specific force generation and is associated with cleavage and/or depletion of key muscle proteins (talin, MHC), effects prevented by muscle-specific calpastatin overexpression. These data indicate that calpain activation is a major cause of diaphragm weakness in response to CLP-induced sepsis.

Renal tuberculosis and iliopsoas abscess: Two case reports.

The urinary system is the second most commonly affected site of extrapulmonary tuberculosis (TB). Due to the diverse and atypical clinical manifestations of urinary TB, the disease is easy to misdiagnose. In the present study, two cases of renal TB are reported, which had completely different clinical manifestations. The first case is a female who presented with loin pain and fever. Purified protein derivative (PPD) and TB antibody tests were negative and computed tomography (CT) scans showed a low density focus in the right kidney with an iliopsoas abscess. The typical CT findings indicated renal tuberculosis. Anti-TB drugs were effective proved the diagnosis. The second case is a male who presented with intermittent gross hematuria. Acid-fast bacilli in urine and TB antibody tests were positive. CT scans revealed a low density focus in the unilateral kidney with a slight expansion of the pelvis, calices and ureter. The patients were treated with the anti-TB drugs and the clinical manifestations disappeared. The diagnosis of urinary TB is challenging in certain cases; when there is no response to the usual antibiotics in patients with fever or gross hematuria, TB should be suspected. CT is the mainstay for investigating possible urinary TB.

Relevance of class 1 integrons and extended-spectrum ß-lactamases in drug-resistant Escherichia coli.

Escherichia coli is a common cause of community­ and hospital­acquired urinary tract infections, and class 1 integrons are the prior elements of gene transference in the capture and distribution of gene cassettes among clinical gram-negative bacillus. In the present study, the resistance of Escherichia coli to antimicrobial agents was investigated. A total of 97 isolates were found to be susceptible to 16 antimicrobial agents and were detected in the production of extended ß­lactamases (ESBLs), distribution of CTX­M­type ß­lactamases, presence and characterization of class 1 integrons and a variable region of integron­positive isolates. Escherichia coli isolates possessing CTX­M (31; 32%) were detected in 19 isolates (61.5%). The presence of ESBLs was associated with resistance to penicillins, third-generation cephalosporins, ciprofloxacin, aminoglycosides and monocyclic ß­lactam antibiotics. Escherichia coli isolates (69; 71.1%) possessed class 1 integrons associated with resistance to ciprofloxacin and numerous third-generation cephalosporins, penicillins, tobramycin and trimethoprim­sulfamethoxazole. The four gene cassette arrangements were as follows: dfrA17­aadA5, aadA1, aacC4­cmlA1 and dfr2d, and 8 carried two disparate class 1 integrons. Five isolates presented class 1 integrons containing no gene cassettes. The distribution of ESBLs and class 1 integrons in Escherichia coli were prevalent with drug resistance in Chengdu. In addition, the resistance range of Escherichia coli isolates that harboured ESBLs and carried class 1 integrons were similar. The current study demonstrated the presence of class 1 integrons and ESBLs, which jointly mediate the resistance of Escherichia coli isolates to a number of antibacterial agents.